• Title/Summary/Keyword: Antifungal protein

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Introduction and Expression of PAP gene using Agrobacterium in Scrophularia buergeriana Miquel (Agrobacterium을 이용한 PAP 유전자의 현삼으로 도입 및 형질발현)

  • Yu, Chang-Yeon;Seong, Eun-Soo;Lim, Jung-Dae;Huang, Shan-Ai;Chae, Young-Am
    • Korean Journal of Medicinal Crop Science
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    • v.9 no.2
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    • pp.156-165
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    • 2001
  • Exogeneous application of pokeweed antiviral protein (PAP), a ribosomal-inacivating protein in the cell wall of Phytolacca americana (pokeweed) protects heterologous plants from viral and fungal infection. A cDNA clone of PAP introduced into Scrophularia buergeriana Miquel by thransformation with Agrobacterium tumefaciences. For plant transformation, explants were precultured on shoot induction medium without kanamycin for 2-5 day, and then they were cocultured with Agrobacterium for 10 minutes. The explants were placed on co culture medium in dark condition, $28^{\circ}C$ for 2days. After explants were washed in MS liquid medium, they were transferred into selection medium including kanamycin 50mg/L (MS salts+1mg/ l BAP+2mg/ l TDZ+0,2mg/ l NAA+MS vitamin+3% sucrose+0.8% agar, pH5.8). From PCR analysis, NPT II band was confirmed in transgenic plant genome and showed resistance against fungi in antifungal activity test. Micro assay to which protein extracted from transgenic line were added, revealed hyphae growth inhibition and no spore germination at high concentration. The characteristics of inhibited hyphae was represented transparent and thin. Expression of PAP in transgenic plants offers the possibility of developing resistance to viral and fungal infection.

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Development of Microbial Inoculant Using By-product of Oriental Herbal Medicine (한약재박을 이용한 미생물제제의 개발)

  • Joo, Gil-Jae;Kim, Young-Mog;Woo, Cheol-Joo;Lee, Oh-Seuk;Kim, Joung-Woong;So, Jae-Hyun;Kwak, Yun-Young;Lee, Jong-Jin;Kim, Jin-Ho;Rhee, In-Koo
    • Applied Biological Chemistry
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    • v.48 no.3
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    • pp.201-206
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    • 2005
  • The development of microbial inoculant was conducted using a by-product of oriental herbal medicine. The constituent of the by-product, which was high in organic matter, was 11.3% of crude protein, 5.1% of crude lipid, 49.7% of NDF (neutral detergent fiber), and 33.8% of ADF (acid detergent fiber). Microorganisms isolated from the by-product of oriental herbal medicine were 35 species. Among them, 6 bacterial species, 4 fungal species, 2 actnomycetes species, and 1 yeast species were effective in the utilization of the by-products. The 13 strains screened were tested for the plant growth-promoting effect in soybean seedling. BL-333 strain was found to increase the soybean yield by about 23% as compared with control. The strain BL-333 was identified as Paenibacillus marcerans. P. marcerans BL-333 showed high anti-fungal activities against virulent fungi, especially Fusarium sp. and Collectotrichum sp. Yields of plants which were inoculated with microbial inoculant prepared with P. marcerans BL-333 and by-product of oriental herbal medicine were found to be higher than control by $3{\sim}24%$. The yield was especially promoted in lettuce, radish, chinese cabbage and cucumber plants.

Cloning and Characterization of a Cellulase Gene from a Plant Growth Promoting Rhizobacterium, Bacillus subtilis AH18 against Phytophthora Blight Disease in Red-Pepper (고추역병을 방제하는 PGPR균주 Bacillus subtilis AH18의 항진균성 Cellulase 유전자의 Cloning 및 효소 특성 조사)

  • Woo, Sang-Min;Jung, Hee-Kyoung;Kim, Sang-Dal
    • Microbiology and Biotechnology Letters
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    • v.34 no.4
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    • pp.311-317
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    • 2006
  • Using PCR amplification, we cloned a cellulase gene (ce/H) from the Bacillus subtilis AH18 which has plant growth-promoting activity and antagonistic ability against pepper blight caused by Phytophthora capsici. The 1.6 kb PCR fragment contained the full sequence of the cellulase gene and the 1,582 bp gene deduced a 508 amino acid sequence. Similarity search in protein database revealed that the cellulase of B. subtilis AH18 was more than 98% homologous in the amino acid sequence to those of several major Bacillus spp. The ce/H was expressed in E. coli under an IPTG inducible lac promoter on the vector, had apparent molecular weight of about 55 kDa upon CMC-SDS-PAGE analysis. Partially purified cellulase had not only cellulolytic activity toward carboxymethyl-cellulose (CMC) but also insoluble cellulose, such as Avicel and filter paper (Whatman No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. The optimum pH and temperature of the ce/H coded cellulase were determined to be pH 5.0 and $50^{\circ}C$. The enzyme activity was activated by $AgNO_3$ or $CoCl_2$. However its activity was Inhibited by $HgC1_2$. The enzyme activity was activated by hydroxy urea or sodium azide and inhibited by CDTA or EDTA. The results indicate that the cellulase gene, ce/H is an antifungal mechanism of B. subtilis AH18 against phytophthora blight disease in red-pepper.

Isolation and Taxonomical Characterization of Strain KM1-15 with Antibiotic Activity from Pine Mushroom (Tricholoma matsutake) Basal Soil (송이 자실체 기저부 토양으로부터 항균활성을 가지는 KM1-15 균주의 분리 및 분류학적 특성)

  • Kim, Yun-Ji;Whang, Kyung-Sook
    • Korean Journal of Microbiology
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    • v.44 no.1
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    • pp.56-62
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    • 2008
  • Two hundred and sixty-eight bacterial strains were isolated from pine mushroom (Tricholoma matsutake) basal soil. In the course of screening for antifungal activity against seven plant pathogenic fungi (Alternaria panax, Botrytis cinerea, Colletotrichum gloeosprioides, Fusarium oxysporum, Phytopthora capsici, Pythium ultimum, Rizoctonia solani) of isolates, strain KM1-15 showed strong antibiotic activity against Alternaria panax and Colletotrichum gloeosprioides. In determining its relationship on the basis of 16S rDNA sequence, KM1-15 strain was most closely related to Bacillus $koguryoae^T$ (AY904033) (99.62%). When assayed with the API 50CHE Kit, unlike Bacillus koguryoae, it is positive for utilization of L-arabinose, cellobiose, inulin, and D-turanose. Results of cellular fatty acid analysis showed that major cellular fatty acids were 15:0 anteiso (35.78%) and 17:0 anteiso (17.97%). In particular, hydroxyl fatty acids such as 13:0 iso 3-OH, 14:0 iso 3-OH, 15:0 iso 3-OH, and 17:0 iso 3-OH were only restricted to strain KM1-15. DNA G+C content was 43.7 mol% and quinone system was MK-7 (100%) in strain KM1-15.

Screening of Biologically Active Compounds from Weeds I (잡초(雜草)에 함유(含有)된 생리활성물질(生理活性物質) 탐색(探索) I)

  • Kim, C.J.;Kang, B.H.;Lee, I.K.;Ryoo, I.J.;Park, D.J.;Lee, K.H.;Lee, H.S.;Yoo, I.D.
    • Korean Journal of Weed Science
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    • v.14 no.1
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    • pp.16-22
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    • 1994
  • Ninty three species of domestic weeds were collected and screened for antimicrobial, antitumor, antioxidant and herbicidal activities. Among them, few showed antifungal activities. Cuscuta japonica showed inhibitory activity against Alternaria mali, Ambrosia artemisiifolia and Geranium sibiricum against Phytophthora capsici, Aster yomema and Aster pilosus against Phytophthora parasitica. Ambrosia artemisiifolia, Artemisia princeps, Artemisia capillaris, Ludwigia prostrata, Chrysanthemum zawadskii, Bidens frondosa, and Geranium sibiricum showed broad antibacterial activities. Carex chordorhiza, Artemisia capillaris, Persicaria nodosa, Senecio koreanus, Pariicum bisulcatum, Geranium sibiricum showed antiblebbing activity on human chronic leukemia K562 cell, among them, Persicaria nodosa was the strongist. Angelica decursiva, Equisetum arvense, Cimicifuga heracleifolia, Persicaria nodosa, Geranium sibiricum, Oenothera odorata, Cyperus sanguinolentus showed antioxidant activities. Ludwigia prostrata and Peucedanum terebinthaceum showed strong herbicidal activities.

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Regeneration of adventitious root from Calystegia soldanella L. in Jeju island and mass proliferation method using bioreactor system (제주지역 갯메꽃(Calystegia soldanella L.) 유래 부정근 재분화 및 생물반응기 시스템 이용 대량증식법)

  • Jong-Du Lee;Eunbi Jang;Weon-Jong Yoon;Yong-Hwan Jung
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2021.04a
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    • pp.37-37
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    • 2021
  • Calystegia soldanella L. is a perennial herbaceous halophyte belonging to the convolvulaceae family, which mainly grows in coastal sand dunes in Korea. Shoots and rhizomes are edible, and roots called 'Hyoseon Chogeun' are known to have medicinal effects such as antipyretic, sterilization, and diuretic. In addition, physiological activities of antioxidant, anti-inflammatory, antiviral, antifungal and PTP-1B (protein tyrosine phosphate-1B) inhibition have been reported. In this study, in vitro induction cell lines of C. soldanella L. collected from the coastal sand dunes in Jeju island was redifferentiated into adventitious roots that can be used as medicinal resources. Also the biomass of mass-proliferated adventitious roots using a bioreactor were evaluated. Plants of C. soldanella L. were collected from the crevice of the seashore in the coastal area of Taeheung 2-ri, Namwon-eup, Seogwipo-si. Then, it was separated into leaves, stems, rhizomes, and roots, and surface sterilized with 70% ethyl alcohol and 2% NaOCl (sodium hypochlorite). After washing with sterilized water, each organ section was cultured in Hormone-free MS medium (Murashige & Skoog Medium). As a result, the induction response rates were evaluated at 85% and 55%, respectively, in terms of callus formation and shoot generation in the rhizome segment. In the case of the adventitious roots morphological characteristics induced by single-use treatment of auxin-based plant growth regulators IBA and NAA from redifferentiated shoots were compared. Most efficient adventitious root culture method as a rooting rate, number, length, and biomass proliferation in the bioreactor system was confirmed when treated by culturing in MS salts, Sucrose 30 g·L-1, and IBA 1mg·L-1 for 4 weeks. In this study, the medium composition and culture period were confirmed using a bioreactor system to mass-proliferate adventitious roots derived from C. soldanella L. in Jeju island. Also this adventitious root line developed a new medicinal material could increase value of the bio-industry ingredient through quantitative and qualitative screening of phyto-bioactive compounds.

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Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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